U.S. patent application number 14/808205 was filed with the patent office on 2016-01-21 for white blood cell analysis system and method.
The applicant listed for this patent is Abbott Laboratories. Invention is credited to Giacomo Vacca, Jiong Wu.
Application Number | 20160018310 14/808205 |
Document ID | / |
Family ID | 47090459 |
Filed Date | 2016-01-21 |
United States Patent
Application |
20160018310 |
Kind Code |
A1 |
Wu; Jiong ; et al. |
January 21, 2016 |
White Blood Cell Analysis System and Method
Abstract
Systems and methods for analyzing blood samples, and more
specifically for performing a white blood cell (WBC) differential
analysis. The systems and methods screen WBCs by means of
fluorescence staining and a fluorescence triggering strategy. As
such, interference from unlysed red blood cells (RBCs) and
fragments of lysed RBCs is substantially eliminated. The systems
and methods also enable development of relatively milder WBC
reagent(s), suitable for assays of samples containing fragile WBCs.
In one embodiment, the systems and methods include: (a) staining a
blood sample with an exclusive cell membrane permeable fluorescent
dye, which corresponds in emission spectrum to an excitation source
of a hematology instrument; (b) using a fluorescence trigger to
screen the blood sample for WB Cs; and (c) using measurements of
(1) axial light loss, (2) intermediate angle scatter, (3)
90.degree. polarized side scatter, (4) 90.degree. depolarized side
scatter, and (5) fluorescence emission to perform a differentiation
analysis.
Inventors: |
Wu; Jiong; (Los Gatos,
CA) ; Vacca; Giacomo; (San Jose, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Abbott Laboratories |
Abbott Park |
IL |
US |
|
|
Family ID: |
47090459 |
Appl. No.: |
14/808205 |
Filed: |
July 24, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13456729 |
Apr 26, 2012 |
9091624 |
|
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14808205 |
|
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61482541 |
May 4, 2011 |
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Current U.S.
Class: |
506/39 |
Current CPC
Class: |
G01N 2015/1477 20130101;
G01N 2021/6439 20130101; G01N 15/1459 20130101; G01N 2015/008
20130101; G01N 2015/1488 20130101; G01N 2015/1006 20130101; G01N
33/49 20130101; G01N 15/1434 20130101; G01N 21/6428 20130101 |
International
Class: |
G01N 15/14 20060101
G01N015/14; G01N 33/49 20060101 G01N033/49; G01N 21/64 20060101
G01N021/64 |
Claims
1. A hematology analyzer for conducting a white blood cell (WBC)
differential analysis on a blood sample that contains a plurality
of WBCs, the analyzer comprising: an excitation source positioned
to excite particles within the blood sample; a plurality of
detectors including (1) an axial light loss detector positioned to
measure axial light loss from the excited blood sample, (2) an
intermediate angle scatter detector positioned to measure
intermediate angle scatter from the excited blood sample, (3) a
polarized side scatter detector positioned to measure 90.degree.
polarized side scatter from the excited blood sample, (4) a
depolarized side scatter detector positioned to measure 90.degree.
depolarized side scatter from the excited blood sample, and (5) a
fluorescence detector positioned to measure fluorescence emitted
from the excited blood sample; and a processor configured to: (a)
dilute the blood sample with a WBC reagent that includes a red
blood cell (RBC) lysing agent and a cell membrane permeable,
nucleic acid binding fluorescent dye; (b) incubate the diluted
blood sample of step (a) for an incubation period of time; (c)
deliver the incubated sample from step (b) to a flow cell in the
hematology analyzer; (d) excite the incubated sample from step (c)
with an excitation source as the incubated sample traverses the
flow cell; (e) collect a plurality of light scatter signals and
fluorescence emission signals from the excited sample; (f) prior to
performing a WBC differential analysis, exclude nuclei-free events
and retain nuclei-containing events using only a fluorescence
trigger that is limited to fluorescence emission signals and is set
to a fluorescence magnitude that is greater than fluorescence
emission signals from RBCs, including RBC fragments, and is less
than fluorescence emission signals from WBCs; and (g) perform the
WBC differential analysis on the nuclei-containing events collected
in step (f).
2. (canceled)
3. The hematology analyzer of claim 1, wherein the axial light loss
detector measures axial light loss at 0.degree. scatter.
4. The hematology analyzer of claim 1, wherein the intermediate
angle scatter detector measures light angle scatter at about
3.degree. to about 15.degree..
5. The hematology analyzer of claim 1, wherein the plurality of
detectors include one or more photomultiplier tubes.
6. The hematology analyzer of claim 1, wherein the excitation
source is a laser.
7. The hematology analyzer of claim 6, wherein the laser is
configured to emit light at a wavelength corresponding to the
fluorescent dye.
8. The hematology analyzer of claim 1, wherein the fluorescent dye
is selected to correspond with the excitation source, and wherein
the fluorescent dye is cell membrane permeable, and nucleic acid
binding.
9. The hematology analyzer of claim 1, further comprising: an
incubation subsystem for diluting the blood sample with a WBC
reagent.
10. The hematology analyzer of claim 9, wherein the WBC reagent
includes the fluorescent dye and a lysing agent.
11. The hematology analyzer of claim 9, wherein the WBC reagent
includes (a) at least one surfactant, (b) at least one buffer or at
least one salt, (c) at least one antimicrobial agent, and (d) the
fluorescent dye.
12. The hematology analyzer of claim 9, wherein the incubation
subsystem is configured to incubate the blood sample with the WBC
reagent for a period of time of less than 25 seconds.
13. The hematology analyzer of claim 9, wherein the incubation
subsystem is configured to incubate the blood sample with the WBC
reagent for a period of time of less than 17 seconds.
14. The hematology analyzer of claim 9, wherein the incubation
subsystem is configured to incubate the blood sample with the WBC
reagent for a period of time of less than 9 seconds.
15. The hematology analyzer of claim 9, wherein the incubation
subsystem is configured to incubate the blood sample with the WBC
reagent at a temperature ranging from 30.degree. C. to 50.degree.
C.
16. The hematology analyzer of claim 9, wherein the incubation
subsystem is configured to incubate the blood sample with the WBC
reagent at a temperature of 40.degree. C.
17-23. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit under 35 U.S.C.
.sctn.119(e) of U.S. Provisional Patent Application No. 61/482,541,
titled, Method For Analyzing White Blood Cells, and filed on May 4,
2011, the entire disclosure of which is incorporated by reference
herein.
[0002] This application is also related to application Ser. No.
______, filed on Apr. 26, 2012, titled "NUCLEATED RED BLOOD CELL
ANALYSIS SYSTEM AND METHOD," with Atty Dkt No: ADDV-017
(11041USO1); and application Ser. No. ______, filed on Apr. 26,
2012, titled "BASOPHIL ANALYSIS SYSTEM AND METHOD," with Atty Dkt
No: ADDV-018 (11042USO1), the entire disclosures of which are
herein incorporated by reference in their entirety.
BACKGROUND
[0003] This invention relates to hematology systems and methods.
More specifically, this invention relates to systems and methods
for analyzing blood samples to identifying, classify, and/or
quantify white blood cells (WBC) and WBC sub-populations in a
sample of blood.
[0004] The development of an accurate and efficient hematology
assay for analysis of WBCs, including counting and classification,
has been a challenge. One reason for the difficulty in developing
an accurate and efficient assay is the relatively low concentration
of WBCs (approximately 0.1% to 0.2%) amongst total blood cells in a
sample. Attempts to engineer advanced methods for analyzing WBCs,
and formulating robust WBC reagent(s), have remained one of the top
priorities in the area of automated hematology analyzers.
[0005] Typically, lysis of red blood cells (RBCs) is required to
eliminate interference from RBCs, and concentrate WBCs, before
counting and classifying WBCs and WBC sub-populations. More
specifically, accurate and efficient WBC analysis requires: (1)
complete lysis of RBCs in less than 30 seconds; (2) the breaking of
large fragments of RBCs into smaller pieces after lysis; and (3)
the preservation of WBCs for accurate counting and proper
classification. If the blood sample is "under-lysed," unlysed RBCs,
even in very small concentrations, interfere with WBC counting and
differential analysis. Similarly, larger fragments of lysed RBCs
can interfere with WBC counting and differential analysis. In
practice, it is difficult to separate unlysed RBCs and/or larger
fragments of lysed RBCs from lymphocytes (the smallest WBCs). If
the blood sample is "over-lysed," the classification of WBCs may be
adversely affected on account of excessive damage to cell membranes
of WBCs.
[0006] In some instances, the difficulty of WBC analysis may be
compounded by the presence of two special types of samples; namely,
samples containing lysis-resistant red blood cells (rstRBCs) and
samples containing fragile lymphocytes. In the case of samples
containing rstRBCs, the WBC count and percentage of lymphocytes are
falsely reported "high," on account of the contribution of
particles other than true lymphocytes, consequently posing a risk
of improper diagnoses and treatments for patients. In the case of
samples containing fragile lymphocytes, damaged lymphocytes may not
show their characteristics in a WBC differential analysis. In
addition, exposed nuclei of WBCs may be counted as nucleated red
blood cells (nRBCs), resulting in a false positive count of nRBCs
in certain assays.
BRIEF SUMMARY
[0007] Provided herein are systems and methods for analyzing blood
samples, and more specifically for performing a white blood cell
(WBC) differential analysis. In general, the systems and methods
disclosed screen WBCs by means of fluorescence staining and a
fluorescence triggering strategy. As such, interference from
unlysed RBCs (e.g., rstRBCs) and RBC fragments is substantially or
completely eliminated, thereby ensuring accurate counting and
differentiation of WBCs and WBC sub-populations. The systems and
methods also enable development of relatively milder WBC
reagent(s), suitable for assays of samples containing fragile
lymphocytes (or other fragile WBCs), including aged samples.
[0008] In one embodiment, for example, the systems and methods
disclosed herein include: (a) staining a blood sample with an
exclusive, cell membrane permeable, fluorescent dye, which
corresponds in emission spectrum to an excitation source of a
hematology instrument; (b) using a fluorescence trigger to screen
the blood sample for WBCs; and (c) using a combination of
measurements of (1) axial light loss, (2) intermediate angle
scatter, (3) 90.degree. polarized side scatter, (4) 90.degree.
depolarized side scatter, and (5) fluorescence emission to perform
a differential analysis.
BRIEF DESCRIPTION OF THE FIGURES
[0009] The accompanying drawings, which are incorporated herein,
form part of the specification. Together with this written
description, the drawings further serve to explain the principles
of, and to enable a person skilled in the relevant art(s), to make
and use the systems and methods presented. In the drawings, like
reference numbers indicate identical or functionally similar
elements.
[0010] FIGS. 1A-1E show histograms of a sample of whole blood,
showing WBCs and residues of RBCs following lysis.
[0011] FIG. 1A is a histogram showing the measurement of an axial
light loss signal.
[0012] FIG. 1B is a histogram showing the measurement of
intermediate angle scatter.
[0013] FIG. 1C is a histogram showing the measurement of 90.degree.
polarized side scatter.
[0014] FIG. 1D is a histogram showing the measurement of 90.degree.
depolarized side scatter.
[0015] FIG. 1E is a histogram showing the measurement of
fluorescence.
[0016] FIG. 2 is a cytogram showing the use of a fluorescent
trigger for eliminating any fragments of RBCs from
consideration.
[0017] FIG. 3 is another cytogram showing the use of a fluorescent
trigger for eliminating any fragments of RBCs from
consideration.
[0018] FIG. 4 is a schematic diagram illustrating a hematology
instrument.
[0019] FIGS. 5A-5J show cytograms of a five-part WBC differential
analysis.
[0020] FIG. 5A is a cytogram depicting axial light loss vs.
intermediate angle scatter.
[0021] FIG. 5B is a cytogram depicting 90.degree. polarized side
scatter vs. axial light loss.
[0022] FIG. 5C is a cytogram depicting 90.degree. polarized side
scatter vs. intermediate angle scatter.
[0023] FIG. 5D is a cytogram depicting 90.degree. depolarized side
scatter vs. axial light loss.
[0024] FIG. 5E is a cytogram depicting 90.degree. depolarized side
scatter vs. intermediate angle scatter.
[0025] FIG. 5F is a cytogram depicting 90.degree. depolarized side
scatter vs. 90.degree. polarized side scatter.
[0026] FIG. 5G is a cytogram depicting fluorescence vs. axial light
loss.
[0027] FIG. 5H is a cytogram depicting fluorescence vs.
intermediate angle scatter.
[0028] FIG. 5I is a cytogram depicting fluorescence vs. 90.degree.
polarized side scatter.
[0029] FIG. 5J is a cytogram depicting fluorescence vs. 90.degree.
depolarized side scatter.
[0030] FIG. 6A is a cytogram illustrating analysis of a sample of
whole blood containing lyse-resistant RBCs, using a traditional
method.
[0031] FIG. 6B is a cytogram showing axial light loss vs.
intermediate angle scatter made by a fluorescence-triggered
hematology analyzer, in accordance with an embodiment
presented.
[0032] FIG. 7A is a cytogram illustrating analysis of an aged
sample of whole blood, using a traditional method.
[0033] FIG. 7B is a cytogram showing axial light loss vs.
intermediate angle scatter made by a fluorescence-triggered
fluorescence-triggered hematology analyzer, in accordance with an
embodiment presented.
DETAILED DESCRIPTION
[0034] Provided herein are systems and methods for analyzing blood
samples, and more specifically for performing a white blood cell
(WBC) differential analysis to identify, classify, and count WBCs
and WBC sub-populations. In general, the systems and methods
disclosed screen WBCs by means of fluorescence staining and a
fluorescence triggering strategy. As such, interference from
unlysed red blood cells (RBCs), such as lysis-resistant red blood
cells (rstRBCs), and RBC fragments is substantially eliminated. The
systems and methods disclosed thereby ensure accurate counting and
differentiation of WBCs and WBC sub-populations. The systems and
methods also enable development of relatively milder WBC
reagent(s), suitable for assays of samples containing fragile
lymphocytes (or other fragile WBCs), including aged samples.
[0035] In one embodiment, for example, the systems and methods
disclosed herein include: (a) staining a blood sample with an
exclusive, cell membrane permeable, fluorescent dye, which
corresponds in emission spectrum to an excitation source of a
hematology instrument; (b) using a fluorescence trigger to screen
the blood sample for WBCs; and (c) using a combination of
measurements of (1) axial light loss, (2) intermediate angle
scatter, (3) 90.degree. polarized side scatter, (4) 90.degree.
depolarized side scatter, and (5) fluorescence emission to perform
a WBC differential analysis.
[0036] As used herein, the expression "fluorescence information"
means data collected from a fluorescence channel of a hematology
analyzer. As used herein, the expression "fluorescence channel"
means a detection device, such as a photomultiplier tube, set at an
appropriate wavelength band for measuring the quantity of
fluorescence emitted from a sample.
(1) Use of Fluorescent Dye(s).
[0037] WBCs contain a relatively high concentration of DNA in their
nuclei. Mature RBCs, however, do not contain DNA. Therefore, a
fluorescent dye is selected to differentiate two classes of blood
cells; namely, the blood cells containing nucleic acids and the
blood cells not containing nucleic acids. The purpose of the dye is
to penetrate into live cells easily, bind DNA with high affinity,
and emit strong fluorescence with adequate Stokes shift when the
dye is excited by an appropriate source of light. The peak
absorption of the dye in the visible band substantially matches the
wavelength of the source of light (within 50 nm of the wavelength
of the source of light, more preferably, within 25 nm of the
wavelength of the source of light), in order to be properly excite
the dye and achieve optimal results.
[0038] The fluorescent dye selected is preferably: 1) capable of
binding nucleic acids, 2) capable of penetrating cell membranes of
WBCs, 3) excitable at a selected wavelength when subjected to a
source of light, 4) emits fluorescence upon excitation by the
source of light, and 5) is biostable and soluble in a liquid. The
dye may be selected from group consisting of: acridine orange, SYBR
11, SYBR Green series dye, hexidium iodide, SYTO 11, SYTO 12, SYTO
13, SYTO 14, SYTO 16, SYTO 21, SYTO RNA Select, SYTO 24, SYTO 25
and any equivalents thereof. The dye is used to "activate" WBCs and
"screen out" unlysed RBCs and fragments of RBCs based on a
fluorescence trigger configured in the hematology analyzer. The dye
is typically present at a concentration of from about 0.1 ng/mL to
about 0.1 mg/mL. While various dyes are available, the dye selected
is generally paired with the excitation source of the hematology
analyzer such that a single exclusive dye is used to stain and
excite fluorescence emission in all WBC sub-populations intended to
be identified, quantified, and/or analyzed. As such, a single
(i.e., exclusive) dye can be used to identify, quantify, and
analyze all the WBC subpopulations at once.
[0039] In one embodiment, a fluorescent dye is provided in a WBC
reagent, with combinations of 1) at least one surfactant, 2) at
least one buffer, 3) at least one salt, and/or 4) at least
antimicrobial agent, in sufficient quantities for carrying out
staining and activating up to 1,000.times.10.sup.3 WBCs per
microliter. The at least one surfactant, such as "TRITON" X-100 or
saponin, is used to destroy the membranes of RBC, and reduce the
sizes of fragments of RBCs. The at least one surfactant is
typically present at a concentration of from about 0.001% to about
5%. The at least one antimicrobial agent, such as those from
"TRIADINE" or "PROCLIN" families, is used to prevent the
contamination of the reagent from microbes. The concentration of
the at least one antimicrobial agent is sufficient to preserve the
reagent for the shelf life required. The at least one buffer, such
as phosphate buffered saline (PBS) or
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), is used
to adjust the pH of reaction mixture for controlling lysis of RBCs
and preserving WBCs. The at least one buffer is typically present
at a concentration of from about 0.01% to about 3%. The pH
typically ranges from about 3 to about 12. The at least one salt,
such as NaCl or Na.sub.2SO.sub.4, is used to adjust the osmolality
to increase the effect of lysing and/or optimize WBC preservation.
The at least one salt may be present at a concentration of from
about 0.01% to about 3%. In certain cases, the at least one buffer
can serve as the at least one salt, or the at least one salt can
serve as the at least one buffer. In general, lower osmolality, or
hypotonicity, is used to accelerate the lysis of RBCs. The
osmolality typically ranges from about 20 to about 250 mOsm.
[0040] Lysis of RBCs can be made to occur at a temperature above
room temperature (e.g., between about 30.degree. C. to about
50.degree. C., such as about 40.degree. C.) over a relatively short
period of time (e.g., less than about 25 seconds, less than about
17 seconds, or even less than about 9 seconds), following mixing of
the sample of blood and the WBC reagent at a ratio of about one
part by volume sample to about 35 parts by volume WBC reagent. The
data for analysis is collected with a plurality of optical channels
and at least one fluorescence channel.
[0041] FIGS. 1A-E show the separation of true WBCs from unlysed
RBCs and RBC fragments, in histograms of collected optical
information and a histogram of fluorescence information. The
histogram in FIG. 1A shows a measurement of axial light loss (ALL).
The histogram in FIG. 1B shows a measurement of intermediate angle
scatter (IAS). The histogram in FIG. 1C shows a measurement of
90.degree. polarized side scatter (PSS). The histogram in FIG. 1D
shows a measurement of 90.degree. depolarized side scatter (DSS).
The histogram in FIG. 1E shows a measurement of fluorescence (FL1).
In the histograms, the horizontal axis indicates the value of the
detection channel (or the names of the channels, i.e., ALL, IAS,
PSS, DSS or FL1). The vertical axis indicates counts of components
of the sample of blood. In the histograms, the lines 100 indicate
residues of RBCs and lines 200 indicate WBCs. As used herein,
"residues of RBCs" is synonymous with "fragments of RBCs." As shown
by comparing FIG. 1E to FIGS. 1A-1D, fluorescence information shows
much better separation between the two groups of particles (i.e.,
WBCs and residues of RBCs) than do any of the optical channels,
thereby facilitating the following analysis.
(2) Use of a Fluorescence Trigger.
[0042] Blood cells emit different magnitudes of fluorescence
signals upon excitation of the fluorescent dye by a source of
light. The differences in magnitude of fluorescence signals arise
from the quantity of nucleic acids, namely DNA, inside the cells.
The greater the quantity of DNA, the greater the likelihood of
higher fluorescence signals. Also, efficacy of penetration of cell
membranes, size of the dye, binding kinetics between the dye and
DNA, affinity between the dye and DNA, and other factors, affect
the fluorescence signals. Mature RBCs emit minimal fluorescence
signals because there is no DNA within mature RBCs. Nucleated red
blood cells (nRBCs) emit very strong fluorescence signals, because
not only is DNA inside nuclei of nRBCs, but also the staining is
easier because membranes of nRBCs are destroyed during the lysis
procedure. Unlysed RBCs or RBC fragments do not emit fluorescence,
although they may emit very weak auto-fluorescence. As shown with
reference to FIG. 1E, the cells that emit much stronger
fluorescence signals are the cells having nuclei, namely, WBCs (and
nRBCs when present).
[0043] As such, the systems and methods presented herein use a
fluorescence trigger for collecting and analyzing WBCs. For
example, a fluorescence trigger, usually set between signals from
RBCs and signals from WBCs, can be used to collect signals from
WBCs separately for further analysis. Two examples of using an FL1
trigger are shown in FIG. 2 and FIG. 3. FIG. 2 is a cytogram
showing the use of a fluorescent trigger for eliminating any
fragments of RBCs (nuclei-free particles) and collecting
nuclei-containing events (e.g., WBCs and/or nRBCs). The fluorescent
dye was acridine orange and the concentration of the fluorescent
dye was 3 .mu.g/mL. The voltage of the fluorescent photomultiplier
tube was set at 350 volts. FIG. 3 is a cytogram showing the use of
a fluorescent trigger for eliminating any fragments of RBCs
(nuclei-free particles) and collecting nuclei-containing events
(e.g., WBCs and/or nRBCs). The fluorescent dye was acridine orange
and the concentration of the fluorescent dye was 0.03 .mu.g/mL. The
voltage of the fluorescent photomultiplier tube was set at 500
volts. In a WBC assay using acridine orange staining (even with
drastically different concentrations of the dyes, i.e., 3 .mu.g/mL
in FIGS. 2 and 0.03 .mu.g/mL in FIG. 3) and a properly set FL1
trigger, only the events above the FL1 trigger are
nuclei-containing events (e.g., WBCs and/or nRBCs, if present) and,
consequently, are captured for further analysis.
(3) Use of a Plurality of Optical Channels and at Least One
Fluorescence Channel for Analysis.
[0044] In one embodiment, the WBC differential analysis is
conducted by means of
[0045] Multiple Angle Polarized Scattering Separation technology
(MAPSS), with enhancement from fluorescence information. At least
one photodiode, or at least one photomultiplier tube, or both at
least one photodiode and at least one photomultiplier tube, are
needed to detect light scattered by each blood cell passing through
a flow cell. Two or more photodiodes are used for measuring ALL
signals, which measure about 0.degree. scatter, and IAS signals,
which measure low angle (e.g., about 3.degree. to about 15.degree.)
scatter. Two or more photomultiplier tubes are used for detecting
90.degree. PSS signals and 90.degree. DSS signals. Additional
photomultiplier tubes are needed for FL1 measurements within
appropriate wavelength range(s), depending on the choice of
wavelength of the source of light. Each event captured on the
system thus exhibits a plurality of dimensions of information, such
as ALL, IAS (one or more channels), PSS, DSS, and fluorescence (one
or more channels). The information from these detection channels is
used for further analysis of blood cells.
[0046] FIG. 4 is a schematic diagram illustrating the illumination
and detection optics of an apparatus suitable for hematology
analysis (including flow cytometry). Referring now to FIG. 4, an
apparatus 10 comprises a source of light 12, a front mirror 14 and
a rear minor 16 for beam bending, a beam expander module 18
containing a first cylindrical lens 20 and a second cylindrical
lens 22, a focusing lens 24, a fine beam adjuster 26, a flow cell
28, a forward scatter lens 30, a bulls-eye detector 32, a first
photomultiplier tube 34, a second photomultiplier tube 36, and a
third photomultiplier tube 38. The bulls-eye detector 32 has an
inner detector 32a for 0.degree. light scatter and an outer
detector 32b for 7.degree. light scatter.
[0047] In the discussion that follows, the source of light is
preferably a laser. However, other sources of light can be used,
such as, for example, lamps (e.g., mercury, xenon). The source of
light 12 can be a vertically polarized air-cooled Coherent Cube
laser, commercially available from Coherent, Inc., Santa Clara,
Calif. Lasers having wavelengths ranging from 350 nm to 700 nm can
be used. Operating conditions for the laser are substantially
similar to those of lasers currently used with "CELL-DYN" automated
hematology analyzers.
[0048] Additional details relating to the flow cell, the lenses,
the focusing lens, the fine-beam adjust mechanism and the laser
focusing lens can be found in U.S. Pat. No. 5,631,165, incorporated
herein by reference, particularly at column 41, line 32 through
column 43, line 11. The forward optical path system shown in FIG. 4
includes a spherical plano-convex lens 30 and a two-element
photo-diode detector 32 located in the back focal plane of the
lens. In this configuration, each point within the two-element
photodiode detector 32 maps to a specific collection angle of light
from cells moving through the flow cell 28. The detector 32 can be
a bulls-eye detector capable of detecting axial light loss (ALL)
and intermediate angle forward scatter (IAS). U.S. Pat. No.
5,631,165 describes various alternatives to this detector at column
43, lines 12-52.
[0049] The first photomultiplier tube 34 (PMT1) measures
depolarized side scatter (DSS). The second photomultiplier tube 36
(PMT2) measures polarized side scatter (PSS), and the third
photomultiplier tube 38 (PMTS) measures fluorescence emission from
440 nm to 680 nm, depending upon the fluorescent dye selected and
the source of light employed. The photomultiplier tube collects
fluorescent signals in a broad range of wavelengths in order to
increase the strength of the signal. Side-scatter and fluorescent
emissions are directed to these photomultiplier tubes by dichroic
beam splitters 40 and 42, which transmit and reflect efficiently at
the required wavelengths to enable efficient detection. U.S. Pat.
No. 5,631,165 describes various additional details relating to the
photomultiplier tubes at column 43, line 53 though column 44, line
4.
[0050] Sensitivity is enhanced at photomultiplier tubes 34, 36, and
38, when measuring fluorescence, by using an immersion collection
system. The immersion collection system is one that optically
couples the first lens 30 to the flow cell 28 by means of a
refractive index matching layer, enabling collection of light over
a wide angle. U.S. Pat. No. 5,631,165 describes various additional
details of this optical system at column 44, lines 5-31.
[0051] The condenser 44 is an optical lens system with aberration
correction sufficient for diffraction limited imaging used in high
resolution microscopy. U.S. Pat. No. 5,631,165 describes various
additional details of this optical system at column 44, lines
32-60.
[0052] The functions of other components shown in FIG. 4, i.e., a
slit 46, a field lens 48, and a second slit 50, are described in
U.S. Pat. No. 5,631,165, at column 44, line 63 through column 45,
line 26. Optical filters 52 or 56 and a polarizer 52 or 56, which
are inserted into the light paths of the photomultiplier tubes to
change the wavelength or the polarization or both the wavelength
and the polarization of the detected light, are also described in
U.S. Pat. No. 5,631,165, at column 44, line 63 through column 45,
line 26. Optical filters that are suitable for use herein include
band-pass filters and long-pass filters.
[0053] The photomultiplier tubes 34, 36, and 38 detect either
side-scatter (light scattered in a cone whose axis is approximately
perpendicular to the incident laser beam) or fluorescence (light
emitted from the cells at a different wavelength from that of the
incident laser beam).
[0054] While select portions of U.S. Pat. No. 5,631,165 are
referenced above, U.S. Pat. No. 5,631,165 is incorporated herein by
reference in its entirety.
[0055] FIGS. 5A-J show an example of an enhanced five-part WBC
differential analysis. Neutrophils (NE), lymphocytes (LY),
monocytes (MO), eosinophils (EO) and basophils (BA) were separated
using MAPSS technology and a FL1 channel. The cytogram in FIG. 5A
shows ALL vs. IAS. The cytogram in FIG. 5B shows 90.degree. PSS vs.
ALL. The cytogram in FIG. 5C shows 90.degree. PSS vs. IAS. The
cytogram in FIG. 5D shows 90.degree. DSS vs. ALL. The cytogram in
FIG. 5E shows 90.degree. DSS vs. IAS. The cytogram in FIG. 5F shows
90.degree. DSS vs. 90.degree. PSS. The cytogram in FIG. 5G shows
FL1 vs. ALL. The cytogram in FIG. 5H shows FL1 vs. IAS. The
cytogram in FIG. 5I shows FL1 vs. 90.degree. PSS. The cytogram in
FIG. 5J shows FL1 vs. 90.degree. DSS.
[0056] In addition to the information collected from the four
traditional MAPSS channels (ALL, IAS, PSS, DSS), the FL1 channel
further distinguishes the cell sub-populations (FIGS. 5G through
5J, inclusive). For the case in which acridine orange is used as
the dye for screening WBCs, basophils show relatively low FL1
signals, and monocytes show relatively high FL1 signals, relative
to other WBC sub-populations, i.e., neutrophils, eosinophils, and
lymphocytes. In the cytograms, dots 510 represent neutrophils, dots
520 represent eosinophils, dots 530 represent lymphocytes, dots 540
represent basophils, and dots 550 represent monocytes. The combined
quantitative information from all optical dimensions and the
fluorescence dimension provides an enhanced, and more reliable,
differential analysis for samples of blood containing WBCs.
[0057] FIG. 6A is a cytogram illustrating analysis of a sample of
whole blood containing rstRBCs, using a traditional method. FIG. 6A
shows a cytogram of ALL vs. IAS, using a commercially available
"CELL-DYN" Sapphire.TM. hematology analyzer. FIG. 6B is a cytogram
showing ALL vs. IAS, using an FL1 trigger-enhanced hematology
analyzer.
[0058] The sample of whole blood was the same as that analyzed in
FIG. 6A. The concentration of the fluorescent dye, acridine orange,
was 3 .mu.g/mL. The results show that the method described herein
is accurate and efficient for the two most challenging cases
mentioned previously. In the traditional method, unlysed RBCs,
i.e., the events appearing in the lower left of the cytogram, were
recognized as lymphocytes, thereby resulting in a higher count of
WBCs and a higher percentage of lymphocytes. FIG. 6B shows the
results of an analysis of WBCs of the sample containing rstRBCs by
the method described herein. In the method described herein, the
analysis of WBCs was accurate because no RBCs or residues of RBCs
were recognized.
[0059] FIG. 7A is a cytogram illustrating analysis of an aged (28
hours old) sample of whole blood, which contains more fragile WBCs,
using a traditional method. FIG. 7A is a cytogram showing ALL vs.
IAS, using a "CELL-DYN" Sapphire.TM. hematology analyzer. FIG. 7B
is a cytogram showing ALL vs. IAS, using an FL1 trigger-enhanced
hematology analyzer. The sample of whole blood was the same as that
analyzed in FIG. 7A. The concentration of the fluorescent dye,
acridine orange, was 3 .mu.g/mL.
[0060] The methods described herein enhances WBC analysis for
hematology analyzers.
[0061] The methods described herein provides a more accurate WBC
count and a more accurate classification of WBC sub-populations,
because the interference from unlysed RBCs and RBC fragments is
substantially eliminated. The use of fluorescence provides further
information to improve differential analysis of WBCs. The methods
described herein shows advantages over traditional methods when
analyzing samples having rstRBCs and samples having fragile
WBCs.
Additional Embodiments
[0062] In one embodiment, there is provided a hematology analyzer
for conducting a
[0063] WBC differential analysis on a blood sample that has been
dyed with a fluorescent dye. The analyzer comprises an excitation
source positioned to excite particles within the blood sample. The
analyzer further comprises a plurality of detectors including: (1)
an axial light loss detector positioned to measure axial light loss
from the excited blood sample, (2) an intermediate angle scatter
detector positioned to measure intermediate angle scatter from the
excited blood sample, (3) a polarized side scatter detector
positioned to measure 90.degree. polarized side scatter from the
excited blood sample, (4) a depolarized side scatter detector
positioned to measure 90.degree. depolarized side scatter from the
excited blood sample, and (5) a fluorescence detector positioned to
measure fluorescence emitted from the excited blood sample. The
analyzer further comprises a processor configured to receive the
measurements of (1) axial light loss, (2) intermediate angle
scatter, (3) 90.degree. polarized side scatter, (4) 90.degree.
depolarized side scatter, and (5) fluorescence from the plurality
of detectors. The processor is also configured to perform a WBC
differential analysis of the blood sample, based on all five
measurements, for particles that emit fluorescence above a
fluorescence threshold. The processor may be further configured to
pre-screen the received measurements to remove from consideration
any particles that do not meet the fluorescence threshold. The
axial light loss detector may measure axial light loss at 0.degree.
scatter. The intermediate angle scatter detector may measure light
angle scatter at about 3.degree. to about 15.degree.. The plurality
of detectors may include one or more photomultiplier tubes. The
excitation source may be a laser configured to emit light at a
wavelength corresponding to the fluorescent dye. Alternatively, the
fluorescent dye may be selected to correspond with the excitation
source. The fluorescent dye may be cell membrane permeable, and
nucleic acid binding.
[0064] The hematology analyzer may further comprise an incubation
subsystem for diluting the blood sample with a WBC reagent. The WBC
reagent may include the fluorescent dye and one or more lysing
agents. Alternatively, the WBC reagent may include (a) at least one
surfactant, (b) at least one buffer or at least one salt, (c) at
least one antimicrobial agent, and (d) the fluorescent dye. The
incubation subsystem may be configured to incubate the blood sample
with the WBC reagent for a period of time of less than about 25
seconds, less than about 17 seconds, or less than about 9 seconds.
The incubation subsystem may also be configured to incubate the
blood sample with the WBC reagent at a temperature ranging from
about 30.degree. C. to about 50.degree. C., such as about
40.degree. C.
[0065] In another embodiment, there is provided a method of
configuring a hematology analyzer to perform a WBC differential
analysis on a blood sample that has been dyed with a fluorescent
dye. The method includes positioning an excitation source to excite
particles within the blood sample. The method further includes
positioning a plurality of detectors to measure (1) axial light
loss, (2) intermediate angle scatter, (3) 90.degree. polarized side
scatter, (4) 90.degree. depolarized side scatter, and (5)
fluorescence from the excited blood sample. The method further
comprises configuring a processor configured to receive the
measurements of (1) axial light loss, (2) intermediate angle
scatter, (3) 90.degree. polarized side scatter, (4) 90.degree.
depolarized side scatter, and (5) fluorescence from the plurality
of detectors. The method also includes configuring the processor to
perform a WBC differential analysis of the blood sample, based on
all five measurements, for particles that emit fluorescence above a
fluorescence threshold. The method may include configuring the
processor to pre-screen the received measurements to remove from
consideration any particles that do not meet the fluorescence
threshold. The axial light loss detector may measure axial light
loss at 0.degree. scatter. The intermediate angle scatter detector
may measure light angle scatter at about 3.degree. to about
15.degree.. The plurality of detectors may include one or more
photomultiplier tubes. The method may also include configuring the
excitation source to emit light at a wavelength corresponding to
the fluorescent dye. Alternatively, the fluorescent dye may be
selected to correspond with the excitation source. The fluorescent
dye may be cell membrane permeable, and nucleic acid binding.
[0066] The method may further comprise configuring an incubation
subsystem of the hematology analyzer to incubate the blood sample
with a WBC reagent. The WBC reagent may include the fluorescent dye
and a lysing agent. Alternatively, the WBC reagent may include (a)
at least one surfactant, (b) at least one buffer or at least one
salt, (c) at least one antimicrobial agent, and (d) the fluorescent
dye. The incubation subsystem may be configured to incubate the
blood sample with the WBC reagent for a period of time of less than
about 25 seconds, less than about 17 seconds, or less than about 9
seconds. The incubation subsystem may also be configured to
incubate the blood sample with the WBC reagent at a temperature
ranging from about 30.degree. C. to about 50.degree. C., such as
about 40.degree. C.
[0067] In another embodiment, there is provided a hematology
analyzer for performing a WBC differential analysis, comprising:
(1) means for exciting particles within a blood sample, which
includes positioning a laser light source to excite the blood
sample as it transverses a flowcell of the hematology analyzer, or
equivalents thereof; (2) means for measuring a plurality of light
scatter signals from the excited particles within the blood sample,
which includes a plurality of detectors (as discussed above), or
equivalents thereof; (3) means for measuring a fluorescence signal
from the excited particles within the blood sample, which includes
fluorescence detectors (as discussed above), or equivalents
thereof; (4) means for screening the excited particles to remove
from consideration any particles that do not meet a fluorescence
threshold, which includes a processor configured with a
fluorescence trigger (as discussed above), or equivalents thereof;
and (5) means for performing a WBC differential analysis, based on
the plurality of light scatter signals and the fluorescence signal,
for the particles passing the means for screening, which includes a
processor configured to perform a WBC differential (as discussed
above), or equivalents thereof. The hematology analyzer may further
comprise means for incubating the blood sample with a WBC reagent
for an incubation period of less than about 25 seconds, which
includes an incubation subsystem (as discussed above), or
equivalents thereof. The hematology analyzer may further comprise
means for incubating the blood sample with a WBC reagent at a
temperature ranging from about 30.degree. C. to about 50.degree.
C., which includes an incubation subsystem (as discussed above), or
equivalents thereof.
[0068] In another embodiment, there is provided a method of
performing a WBC analysis with an automated hematology analyzer.
The method comprises: (a) diluting a sample of whole blood with a
WBC reagent, wherein the WBC reagent includes a RBCs lysing agent
and a fluorescent dye that penetrates WBC membranes and binds to
WBC nucleic acids; (b) incubating the diluted blood sample of step
(a) for an incubation period of less than about 25 seconds, at a
temperature ranging from about 30.degree. C. to about 50.degree.
C.; (c) delivering the incubated sample from step (b) to a flow
cell in the hematology analyzer; (d) exciting the incubated sample
from step (c) with an excitation source as the incubated sample
traverses the flow cell; (e) collecting a plurality of light
scatter signals and a fluorescence emission signal from the excited
sample; and (0 performing a WBC differential based on all the
signals collected in step (e), while removing from consideration
any particles within the diluted blood sample that do not meet a
fluorescence threshold based on the fluorescence emission signal.
The WBC reagent may include (a) at least one surfactant, (b) at
least one buffer or at least one salt, (c) at least one
antimicrobial agent, and (d) at least one fluorescent dye. The
excitation source may have a wavelength of from about 350 nm to
about 700 nm. The fluorescence emission may be collected at a
wavelength of from about 360 nm to about 750 nm, by a band-pass
filter or a long-pass filter.
[0069] In yet another embodiment, there is provided a method for
counting and classifying WBCs by means of an automated hematology
analyzer, the method comprising the steps of: (a) diluting a sample
of whole blood with at least one WBC reagent; (b) incubating the
diluted sample of step (a) for sufficient period of time within a
selected temperature range to lyse RBCs, to preserve WBCs, to allow
at least one fluorescent dye to penetrate cell membranes of the
WBCs, and bind nucleic acids within the nuclei of the WBCs; (c)
delivering the incubated sample from step (b) to a flow cell in a
stream; (d) exciting the incubated sample from step (c) by means of
a source of light as the incubated sample traverses the flow cell;
(e) collecting a plurality of optical scatter signals and at least
one fluorescence emission signal simultaneously; and (f)
differentiating and quantifying WBCs by means of the signals
collected in step (e). Features of the embodiment include, but are
not limited to: (1) use of at least one fluorescent dye to bind and
stain nucleic acids in WBCs and other nuclei-containing cells in a
given sample of blood during the procedure for lysing RBCs, and to
induce fluorescent emissions upon excitation by photons from a
given source of light, such as a laser beam at an appropriate
wavelength; (2) use of a fluorescent trigger to separate and
collect events that emit strong fluorescence (e.g., events
involving WBCs and other nuclei-containing cells); (3) use of a
plurality of optical channels and at least one channel for
fluorescence for collecting data and analyzing the data so
collected in order to identify each cell population and reveal
additional information.
[0070] In yet another embodiment, the systems and methods disclosed
herein include: (a) staining a blood sample with an exclusive, cell
membrane permeable, fluorescent dye, which corresponds in emission
spectrum to an excitation source of a hematology instrument; (b)
using a fluorescence trigger to screen the blood sample for WBCs;
and (c) using a combination of measurements to perform a
differential analysis. The combination of measurements may include
one or more measurements selected from the group consisting of:
axial light loss, intermediate angle scatter, 90.degree. polarized
side scatter, 90.degree. depolarized side scatter, one or more
fluorescence emission measurements, multiple ring intermediate
angle scatter, and any combinations or equivalents thereof.
[0071] In one embodiment, the invention is directed toward one or
more computer systems capable of carrying out the functionality
described herein. For example, any of the method/analysis steps
discussed herein may be implemented in a computer system having one
or more processors, a data communication infrastructure (e.g., a
communications bus, cross-over bar, or network), a display
interface, and/or a storage or memory unit. The storage or memory
unit may include computer-readable storage medium with instructions
(e.g., control logic or software) that, when executed, cause the
processor(s) to perform one or more of the functions described
herein. The terms "computer-readable storage medium," "computer
program medium," and "computer usable medium" are used to generally
refer to media such as a removable storage drive, removable storage
units, data transmitted via a communications interface, and/or a
hard disk installed in a hard disk drive. Such computer program
products provide computer software, instructions, and/or data to a
computer system, which also serve to transform the computer system
from a general purpose computer into a special purpose computer
programmed to perform the particular functions described herein.
Where appropriate, the processor, associated components, and
equivalent systems and sub-systems thus serve as examples of "means
for" performing select operations and functions. Such "means for"
performing select operations and functions also serve to transform
a general purpose computer into a special purpose computer
programmed to perform said select operations and functions.
Conclusion
[0072] The foregoing description of the invention has been
presented for purposes of illustration and description. It is not
intended to be exhaustive or to limit the invention to the precise
form disclosed. Other modifications and variations may be possible
in light of the above teachings. The embodiments were chosen and
described in order to best explain the principles of the invention
and its practical application, and to thereby enable others skilled
in the art to best utilize the invention in various embodiments and
various modifications as are suited to the particular use
contemplated. It is intended that the appended claims be construed
to include other alternative embodiments of the invention;
including equivalent structures, components, methods, and
means.
[0073] The above Detailed Description refers to the accompanying
drawings that illustrate one or more exemplary embodiments. Other
embodiments are possible. Modifications may be made to the
embodiment described without departing from the spirit and scope of
the present invention. Therefore, the Detailed Description is not
meant to be limiting. Further, the Summary and Abstract sections
may set forth one or more, but not all exemplary embodiments of the
present invention as contemplated by the inventor(s), and thus, are
not intended to limit the present invention and the appended claims
in any way.
* * * * *